The long-term goal of this project is to determine the role of membrane lipid composition in the regulation of endothelial ion channels. Our recent studies have shown that two major types of endothelial ion channels, the inwardly-rectifying K+ channels (Kir) and volume-regulated CI- channels (VRAC) are strongly suppressed by the elevation of cellular cholesterol, a major risk factor for the development of atherosclerosis. This study focuses on Kir channels that play the dominant role in setting endothelial membrane potential under static and hemodynamic conditions and are also known to play an important role in the regulation of endothelium-mediated control of vascular tone. In preliminary studies, we have shown that an increase in cellular cholesterol results in a decrease in the number of active Kir channels. To understand the mechanisms underlying this effect, we propose: (1) To define the structural determinants of the Kir channel that are responsible for this effect. Specifically, to determine whether cholesterol-induced suppression of Kir is mediated by intracellular signaling pathways that are known to regulate Kir, and whether the sensitivity of the Kir channels to cholesterol is mediated by the cytosolic tails or by the transmembrane domains of the channels. Discrimination between the two will give a strong indication of whether the interaction between cholesterol and Kir is direct or mediated by other proteins. Structural analysis will be performed using two complementary strategies: comparative analysis of different Kirs and chimeric proteins, and site-directed mutagenesis. (2) To determine whether a decrease in the number of active Kir channels is due to the suppression of Kir channel protein expression, disruption of its targeting to the plasma membrane or abnormal partition into cholesterol-rich lipid domains. Western blot analysis, quantitative PCR, 3D imaging of fluorescently-tagged Kir proteins and isolation of cholesterol-rich lipid domains will be utilized. (3) To determine whether an increase in cellular cholesterol suppresses Kir channels exposed to physiological levels of shear stress. The effect of cholesterol on Kir activity will be evaluated under well-defined shear stress conditions using a novel "Minimally Invasive Flow" device that was developed for this purpose.